Beta-lactamases are enzymes which are synthesized by a large number of bacterial species, but not by mammalian tissues. The detection of beta-lactamase in a mammalian body fluid is often indicative of a bacterial infection. Although the beta-lactamases synthesized by different bacteria vary in their structure and in certain properties, all are capable of hydrolyzing the carbon-nitrogen bond of the beta-lactam ring of a penicillin or a cephalosporin. Thus, penicillin and cephalosporins have been studied and utilized as a means for detecting the presence of beta-lactamase enzymes. Ross et al., "Beta-Lactamase Assays," Methods for the Study of Antibiotics (1975), pages 69-85; Lucas, T. J., "An Evaluation of 12 Methods for the Demonstration of Penicillinase," J. of Clinical Pathology (1979), pages 1061-1065.
Previous means for detection of beta-lactamases have typically used colorimetric tests, such as iodometric assay to measure the amount of iodine needed to oxidize the product of the enzyme. Sykes et al., "Microiodometric Determination of Beta-Lactamase Activity," Antimicrob. Agents and Chemotherapy, February 1972, pp. 94-99. Other diagnostic tests used particular cephalosporin compounds which undergo a distinctive color change when hydrolyzed by beta-lactamases. O'Callaghan et al., "Novel Method for Detection of Beta-Lactamase by Using a Chromogenic Cephalosporin Substrate," Antimicrob. Agents and Chemotherapy (April 1972), pp. 283-288; Jones, "In Vitro Evaluation of Pyridine-2-Argo-p-Dimethylaniline Cephalosporin, A New Diagnostic Chromogenic Reagent, and Comparison with Nitrocefin, Cephacetrile, and Other Beta-Lactam Compounds," J. of Clinical Microbiology, April 1982, pp. 677-683; Ross et al., supra; and T. J. Lucas, supra. Carbon 14-labeled benzyl-penicillin has been used to detect the presence of beta-lactamases. Yolken et al., "Rapid Diagnosis of Infections Caused by Beta-Lactamase-Producing Bacteria by Means of an Enzyme Radioisotopic Assay," J. of Pediatrics (November 1980), pages 715-720. In this assay, C.sup.14 -penicillin and the sample were mixed together, then an aliquot transferred to a column packed with DEAE-Sephacel equilibrated with tris buffer. The beta-lactamase converted the penicillin to penicillinoic acid, resulting in a new carboxyl group with an increased affinity to a positively charged gel such as DEAE-Sephacel. The unreacted penicillinase was washed from the column, and the penicillinoic acid subsequently recovered and measured.
Recently, beta-lactamases have been used in enzyme immunoassays. Yolken et al., "The Use of Beta-Lactamase in Enzyme Immunoassays for Detection of Microbial Antigens," J. of Immunological Methods (1984), pp. 109-123.
A cephalosporin is any of a family of antibiotics related to penicillin. The cephalosporin molecule contains a fused beta-lactam-dihydrothiazine ring system, typically with an N-acyl side chain at the 7-position and a group attached to the dihydrothiazine ring at the 3-position. Substituents of cephalosporins than can accept an electron in the 3-position, readily leave the compound upon the hydrolysis of the beta-lactam ring. Thus, whenever a cephalosporin is hydrolyzed by a beta-lactamase, there is a concomitant qualitative release of the electron-accepting substituent at the 3-position. O'Callaghan et al., "Correlation between Hydrolysis of the Beta-Lactam Bond of the Cephalosporin Nucleus and Expulsion of the 3-Substituent," J. of Bacteriology (June 1972), pp. 988-991.
None of the aforementioned methods or assays for detecting the presence of beta-lactamases use a cephalosporin immobilized on a solid phase support comprising a beta-lactamase releasable, detectably labeled, substituent at the 3-position thereof.